Noncanonical formation of SNX5 gene-derived circular RNA regulates cancer growth

Oral squamous cell carcinoma (OSCC) is a prevalent cancer worldwide, exhibiting unique regional prevalence. Despite advancements in diagnostics and therapy, the 5-year survival rate for patients has seen limited improvement. A deeper understanding of OSCC pathogenesis, especially its molecular underpinnings, is essential for improving detection, prevention, and treatment. In this context, noncoding RNAs, such as circular RNAs (circRNAs), have gained recognition as crucial regulators and potential biomarkers in OSCC progression. Our study highlights the discovery of previously uncharacterized circRNAs, including a SNX5 gene-derived circRNA, circSNX5, through deep sequencing of OSCC patient tissue transcriptomes. We established circSNX5’s tumor-specific expression and its strong correlation with patient survival using structure-specific and quantitative PCR analyses. In vitro and in vivo experiments underscored circSNX5 RNA’s regulatory role in cancer growth and metastasis. Further, our omics profiling and functional assays revealed that ADAM10 is a critical effector in circSNX5-mediated cancer progression, with circSNX5 maintaining ADAM10 expression by sponging miR-323. This novel circRNA-miRNA-mRNA regulatory axis significantly contributes to oral cancer progression and malignancy. Moreover, we discovered that circSNX5 RNA is produced via noncanonical sequential back-splicing of pre-mRNA, a process negatively regulated by the RNA-binding protein STAU1. This finding adds a new dimension to our understanding of exonic circRNA biogenesis in the eukaryotic transcriptome. Collectively, our findings offer a detailed mechanistic dissection and functional interpretation of a novel circRNA, shedding light on the role of the noncoding transcriptome in cancer biology and potentially paving the way for innovative therapeutic strategies.


Figure S1 .
Figure S1.CircSNX5 RNA was expressed in cancer cell lines.(A) RNA-sequencing read counts corresponding to the indicated back-splicing event, with the annotated chromosomal location shown at the bottom.(B) CircSNX5 RNA expression of oral cancer cell lines were analyzed by RT-qPCR experiment and presented by the ΔCq method.(C) Genomic DNA (DNA) and reverse-transcribed cDNA (RNA) of the indicated cells were subjected to PCR assays with convergent (Con) and divergent (Div) primers, and the resulting PCR products were analyzed by gel electrophoresis.(D) HeLa cells were treated with actinomycin D (AD) for the indicated time lengths, and total RNA of treated cells at time-points were collected and subjected to RT-qPCR assays.RNA turnover rate was measured by normalization of RNA abundance to the initial time-point and plotted for individual gene.(E) RNC-mRNA fraction was isolated from cytosolic fraction of the cells, and the indicated gene expression in fractions were determined by RT-qPCR assay.Indicated gene abundance in RNC fraction was calculated by ΔCq method, and normalized to the values of cytosolic fractions.

Figure S2 .Figure S3 .
Figure S2.Parental SNX5 mRNA level remains invariable at circSNX5 knockdown.(A) CircSNX5 RNA expression of control and knockdown SCC25 cells was analyzed by RT-qPCR experiment.(B) Parental SNX5 gene expression of control and circSNX5 knockdown cells were detected by RT-qPCR assays, and the proximal and distal region primers corresponding to backsplicing junction site were used.

Figure S6 .Figure S7 .
Figure S6.Noncanonical sequential back-splicing takes places in the circSNX5 formation.(A) PCR products in Fig 3D were purified and analyzed by the Sanger method, and the results were shown in sequence histograms.The region on the SNX5 gene was annotated and highlighted.(B) Mature and precursor circSNX5 RNA expression of the indicated cells in Fig 3E were analyzed by RT-qPCR.(C) Indicated circSNX5 RNA levels of the subcellular fractions in Fig 3F were measured by RT-qPCR experiments.(D) Cells were treated with actinomycin (ActD) at the indicated time points, and the total RNA of treated cells was harvested and transferred into cDNA.CircSNX5 PCR assays of cDNA samples were performed and visualized by gel electrophoresis, and the PCR amplicons were annotated.ACTIN expression served as the loading control.

Figure S8 .
Figure S8.CircSNX5 overexpression enhances the colony formation and cell migration abilities of oral cancer cell lines.(A) Colony formation assays of control and circSNX5 overexpression SCC25 cells were performed and visualized by crystal violet staining, and the quantification of colony forming was depicted in the bar graph.(B) The wound healing migration assay was performed to assess the migratory ability of SAS cells upon circSNX5 overexpression.Representative images of the culture at the indicated time points after scratching are shown.

Figure S10 .
Figure S9.STAU1 knockdown facilitates the circSNX5 formation.(A) Control and ADAR1specific siRNAs were transfected into cells, and the indicated gene expression of the transfectants was analyzed by RT-qPCR assays.(B) STAU1 RNA levels of the indicated cells were measured by RT-qPCR.(C) Precursor and mature circSNX5 RNA levels of the transfectants in Fig 5B were measured by RT-qPCR.(D) CircSNX5 PCR assays of the indicated samples were performed, and the resulting PCR products were resolved by gel electrophoresis.PCR amplicons were noted and quantified.

Figure S11 .
Figure S11.Omics profiles of the circSNX5 knockdown cells.(A) Differentially expressed genes revealed by the RNA-sequencing assay were subjected to pathway analysis by the DAVID bioinformatics, and the annotated pathways were ranked and shown.(B) The intersection of the differentially expressed gene sets from the proteomic and transcriptomic profiling was performed to explore the downstream target gene network.
Figure S12.Ectopic ADAM10 expression moderates the inhibited phenotypes by circSNX5 knockdown.(A) RT-qPCR results for circSNX5 and ADAM10 RNA expression in the OSCC cohort were subjected to a co-expression analysis (n = 48), and the R-squared value and P-value were calculated and noted.(B) Indicated RNA expression in the transfected SCC25 cells was determined by RT-qPCR.(C) ADAM10 protein expression in the indicated transfected SCC25 cells was measured by Western blot.GAPDH expression served as the loading control.(D and E) Cell proliferation and colony formation ability of the indicated transfectants of SCC25 cells were analyzed by MTT assay and crystal violet staining, respectively.Colony images and quantified results are shown.(F) The mouse xenograft experiment was performed by inoculating the indicated transfected SAS cell lines.Tumors formed at the indicated time points were dissected and measured for volume (left panel).The photographs of mice bearing tumors and the weight of resected tumors are shown.
Figure S13.CircSNX5 RNA functions as a miRNA sponge to regulate oral cancer progression.(A) MiR-323 RNA levels in the transfectants in Fig 6H were collected and subjected to RT-qPCR.(B) MiR-323 and circSNX5 RNA expression in the transfectants in Fig 6I were determined by RT-qPCR.(C) ADAM10 RNA expression in the indicated transfected cells was analyzed by RT-qPC.(D) Pulldown assay was performed with the biotinylated circSNX5-specific probe and control oligo, and the enrichment of miR-323 in the pull-down complex was analyzed by RT-qPCR.(E) Biotin-labeled miR-323 was delivered into SAS cells, and the transfected cells were subjected to a pull-down assay.The enrichment of circSNX5 RNA in the complex was analyzed by RT-qPCR.

Figure S15 .Figure S16 .
Figure S15.CircSNX5 expression neutralizes miR-323-induced downregulation of cell metastasis.(A and B) Transwell migration and invasion assays of the indicated transfectant SAS cells were performed.The representative photographs for the indicated groups were shown in the left panel, and the migration and invasion capacities were quantified and shown in bar graphs.

Table S2 . Proteomic analyses of circSNX5 knockdown cells
Please find the attached excel file.